Background & Aims
Pain is reported more frequently in women than men, and the past decade has witnessed an explosion of research on the relationship between sex and pain [1,2], particularly with respect to estrogens and estrogen receptors [3,4]. By contrast, only a few studies have studied the contribution of testosterone and its cognate androgen receptor (AR) to pain [5–9]. In a mouse model of chronic postsurgical pain (CPSP), we reported that intrathecal administration of the protein kinase A (PKA) inhibitor H89 prevented kappa opioid receptor (KOR) antagonist-induced reinstatement of mechanical hypersensitivity in male but not female mice [10]. These data indicate that the intracellular signaling of latent postsurgical pain sensitization (LS) is driven by PKA signaling in the dorsal horn of male but not female mice. A major gap is the underlying mechanism of this sex difference. To address this question, we tested the hypothesis that ARs activate PKA to drive CPSP.
Methods
In C57BL/6 mice (7-8 wk), we incised the skin and underlying plantaris muscle at the left hindpaw, and then waited 3 wk for hypersensitivity (von Frey filaments) to resolve. We then pharmacologically or surgically activated or inhibited ARs with 4 experiments: 1) in female mice, we administered testosterone enanthate (TE, 0.45 mg/kg, subcutaneous) or vehicle (sesame oil) for two weeks (n = 5-8); 2) in female mice, we spinally administered TE (10 µg, intrathecal) or vehicle (cremophore: ethanol: saline – 1:1:8) (n = 6-8); 3) in male mice, we administered flutamide (50 mg/kg, subcutaneous) to block AR (n = 6-12); 4) male mice received orchiectomy (surgical removal of testes) or sham surgery (n = 6-8). After each of these interventions, we administered vehicle, PKA inhibitor H89 (10 nmol, i.t.), and/or KOR antagonist LY2456302 (10 µg, i.t.) in a 2×2 design in both male and female mice.
Results
Neither vehicle nor H89 alone changed mechanical threshold in any group. LY2456302 reinstated mechanical hypersensitivity in all 4 groups, consistent with previous studies indicating that incision causes concomitant latent postsurgical pain sensitization that is masked by endogenous KOR signaling [10]. In female mice that received systemic (Drug × Time: F(21,129) = 4.480; p < 0.0001) or intrathecal (Drug × Time: F(21,114) = 2.959; p = 0.0001) TE, preadministration of H89 prevented LY2456302-induced reinstatement of mechanical hypersensitivity . On the other hand, in male mice that received subcutaneous AR blocker (Drug × Time: F(21,192) = 3.484; p < 0.0001) or orchiectomy (Drug × Time: F(21,144) = 3.656; p < 0.0001), preadministration of H89 failed to prevent LY2456302-induced reinstatement of mechanical hypersensitivity.
Conclusions
We found that pharmacological or surgical interruption of AR prevented the ability of a PKA inhibitor to prevent pain reinstatement in male mice, whereas pharmacological activation of AR recruited the ability of PKA to prevent reinstatement in female mice. These data show that endogenous testosterone facilitates the PKA signaling component of LS, and that this can be mimicked in females with exogenous testosterone. This could explain why H89 prevents KOR antagonist-induced reinstatement of hypersensitivity in male but not female mice. These data promote ARs as a target for the development of novel analgesics for CPSP, particularly in men. Current studies are using the Cre-lox system, pharmacological, and surgical approaches to determine whether ARs located on sensory or spinal neurons are responsible for activating PKA in CPSP. Furthermore, we will evaluate the effects of estradiol in PKA-mediated CPSP since testosterone can be converted to estradiol by aromatase.
References
1. Fillingim RB, King CD, Ribeiro-Dasilva MC, Rahim-Williams B, Riley JL. Sex, gender, and pain: a review of recent clinical and experimental findings. Journal of Pain. 2009; 10:447–485.
2. Mogil JS. Sex differences in pain and pain inhibition: multiple explanations of a controversial phenomenon. Nature Reviews Neuroscience. 2012; 13:859–866.
3. Paredes S, Cantillo S, Candido KD, Knezevic NN. An association of serotonin with pain disorders and its modulation by estrogens. International Journal of Molecular Sciences. 2019;20.
4. Chen Q, Zhang W, Sadana N, Chen X. Estrogen receptors in pain modulation: cellular signaling. Biology of Sex Differences. 2021;12.
5. Khalilzadeh E, Aliyoldashi M, Abdkarimi B, Azarpey F, Vafaei Saiah G, Hazrati R, et al. Reversal of cold intolerance by testosterone in orchiectomized mice after tibial nerve transection. Behavioural Brain Research. 2023; 441:114269.
6. Sorge RE, LaCroix-Fralish ML, Tuttle AH, Sotocinal SG, Austin JS, Ritchie J, et al. Spinal cord toll-like receptor 4 mediates inflammatory and neuropathic hypersensitivity in male but not female mice. Journal of Neuroscience. 2011; 31:15450–15454.
7. Lesnak JB, Nakhla DS, Plumb AN, McMillan A, Saha S, Gupta N, et al. Selective androgen receptor modulator microparticle formulation reverses muscle hyperalgesia in a mouse model of widespread muscle pain. Pain. 2023; 164:1512–1523.
8. Lesnak JB, Fahrion A, Helton A, Rasmussen L, Andrew M, Cunard S, et al. Resistance training protects against muscle pain through activation of androgen receptors in male and female mice. Pain. 2022; 163:1879–1891.
9. Barbosa Neto JO, Garcia JBS, Cartágenes MDSDS, Amaral AG, Onuchic LF, Ashmawi HA. Influence of androgenic blockade with flutamide on pain behaviour and expression of the genes that encode the NaV1.7 and NaV1.8 voltage-dependent sodium channels in a rat model of postoperative pain. Journal of Translational Medicine. 2019; 17:287.
10. Basu P, Custodio-Patsey L, Prasoon P, Smith BN, Taylor BK. Sex differences in protein kinase A signaling of the latent postoperative pain sensitization that is masked by kappa opioid receptors in the spinal cord. Journal of Neuroscience. 2021; 41:9827–9843.
11. Zimmermann, Manfred. Ethical guidelines for investigations of experimental pain in conscious animals. Pain. 1983; 16: 109–10.